Biotechnology- Antisense Technology

Antisense Technology, Ribozyme

1. gggdfdf WELCOME

2. PRINCIPLES OF BIOTECHNOLOGY MBB-501 Presented by submitted to Vivek suthediya Mr.R.S. deshpande Reg. no. Adpm/19/2680

3. CONTENTS Introduction History Mechanism Approaches Application Conclusion References

4. Antisense Technology The tool that is used for the inhibition of gene expression is called Antisense technology. The antisense technology uses following agents for inhibition of gene expression. I ) Antisense Oligonucleotides II ) Ribozymes III) Short Interfering RNA (siRNA) IV) Micro RNA (miRNA)

5. X Antisense Technology

6. Antisense Oligo-Nucleotides Introduction : Antisense oligonucleotides is a single stranded RNA that is complementary to a messenger RNA (mRNA) strand transcribed in the cell. Antisense oligonucleotides introduced into a cell to inhibit translation of a complementary mRNA by base pairing to it and creating barrier to the translation machinery. This translation arrest causes reduced amount of protein expression. Well known examples of GM plants produced by this technology : The Flavr Savr tomato, Two cultivars of ring spot resistant papaya.

7. History : First time at “Free University of Amsterdam”, used antisense RNA technology against the gene determining flower color of Petunia. Antisense effect first demonstrated by Zemencnick and Stephenson in 1970 on “Robus sarcoma virus”. First time antisense oligonucleotides are synthesized by Eckstein and colleagues.

8. Fig. General Outline

9. Sense Strand : It is the strand that is not transcribed into RNA. The base sequence of sense strand of a gene is the same as that of the mRNA produced by it ( except for T in the place of U ). Hence, the nhRNA/mRNA produced by a gene in normal orientation is also known as sense RNA. It is oriented in 5’→3’ orientation.

10. Antisense Strand : It is a strand that is transcribed into RNA. Its sequence is complementary to the mRNA sequence . The antisense strand is also referred to as the template strand. The oligonucleotide complementary to the mRNA is called ‘antisense’ oligonucleotide. It is oriented in 3’→5’ direction.

11. Fig. Sense and Antisense Strand

12. Mechanism : In this technique, short segments of single stranded RNA are introduced. These oligonucleotides are complementary to the mRNA, which physically bind to the mRNA. So, they block the expression of a particular gene. Antisense oligonucleotides usually consists of 15-20 nucleotides. Translation of mRNA may be blocked by two possible mechanisms : I ) By base specific hybridization, which prevents translation of mRNA.

13. II ) By forming RNA/DNA duplex which is recognized by nuclease RNaseH, specific for digesting RNA in an RNA/DNA duplex. RNaseH is a non-specific endonuclease, catalyzes the cleavage of RNA via hydrolytic mechanism. RNaseH has ribonuclease activity cleaves the 3’-O-P bond of RNA in a DNA/RNA duplex.

14. Characteristics of Antisense Oligonucleotides : Unique DNA sequence Efficient cellular uptake Minimal nonspecific binding Target specific hybridization Non-toxic antisense construct

15. Approaches : The antisense technology can be modified in three modes because of chemical modifications of the oligonucleotides. These modes are due to activation of RNaseH and internucleotides linkages which do not activate enzyme.

16. 1 st Approach : The antisense oligonucleotudes binds the target sequence causing both “hybridization arrest” and “RNaseH activation”. Degradation of mRNA by RNaseH results into release of oligonuleotides. They may bind to other copies of target mRNA. These oligonucleotides are also susceptible to other nucleases. This a major parameter affecting catalytic mode of degradation.

17. 2 nd Approach : In this, antisense oligonucleotides binds to target sequence result in translation arrest but they do not activate enzyme RNaseH. Oligoribonucleotides and analogues, oligodeoxyribonucleotides, various non phosphate and phosphate internucleotides linkages fall in this category. They show resistance against nucleases enzyme and never get degraded by them. They also show effective translation arrest. But the major problem is that they are generally required in higher molar concentrations than those which activate RNaseH.

18. 3 rd Approach : It combines features of both previous approaches. They contain both internucleotides linkages which are responsible for RNaseH activation and which shows resistance against them. Digestion of mRNA target in RNA duplex releases oligonucleotides which are resistant against nuclease enzyme, hence are more effective than oligonucleotides in 1 st approach. They may form hybrids of oligodeoxyribonucleotides and oligoribonucleotides.

19. Ribozyme A ribozyme is an RNA molecule, which has enzymatic activity usually concerned with RNA degradation. In the ribozyme approach a DNA sequence specifying an enzymatic RNA sequence is fused with a sequence of the gene against which the ribozyme is aimed. Therefore the RNA product of this gene construct has a sequence complementary to the sense RNA (mRNA) produced by target gene. The complementary sequence of this RNA pairs with the sense RNA produced by the target gene and the ribozyme sequence linked to it degrades the sense RNA.

20. It degrades the mRNA by cleaving the phosphodiester backbone at a specific cutting site. Types of Ribozymes : RNase P Hammerhead Ribozyme Hairpin ribozyme Group I and group II intron splicing ribozymes.

21. Application of Antisense Technology : In agriculture : Slow fruit softening tomato Changed fatty acid composition of Brassica Oil Delayed senescence in Carnation Male sterility In medicine : Cancer Chemotherapy AIDS therapy Genetic disorders New drug discovery

22. Flavr Savr Tomato : It is developed by Californian company Calgene. The genetically engineered Flavr Savr tomato was introduced on 21 May, 1994. Development of Flavr Savr tomato : Softening of fruit is largely due to dehydration of cell wall (pectin) by enzyme polygalacturonase (PG). The gene encoding PG has been isolated and cloned (pTOM6).

23. Procedure involves : Isolation of DNA from tomato plant that encodes the enzyme polygalacturonase (PG). Transfer of PG gene to a vector bacteria and production of complementary DNA (cDNA) . Introduction of cDNA into a fresh tomato plant to produce transgenic plant. Mechanism : In normal plants, PG gene encodes a normal or sense mRNA that produce the enzyme PG and it is involve in fruit ripening. The cDNA of PG encodes for antisense mRNA, which is complementary to sense RNA. The hybridization between sense and antisense mRNA renders the sense mRNA ineffective.

24. Consequently no polygalacturonase is produced hence fruit ripening is delayed.

25. Role in Drug Discovery : In recent years, Antisense oligonucleotides (AS-OD) technology have been widely used as potent and promising tool for drug discovery and development. Diseases are connected to insufficient or excess production of certain proteins. If the production of these proteins is interrupted i.e. increased or decreased then certain diseases can be cured . The vast majority of drugs available today either act at the protein level, or the drugs themselves are proteins.

26. CONCLUSION

27. Reference : Plant Biotechnology – B.D. Singh Agricultural Biotechnology – Purohit www.sciencedirect.com www.ukessys.com

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